Laboratory 13 Blood Groups, Blood Typing and Blood Transfusions Objectives: •
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Explain the role of surface antigens on RBCs in determining blood groups. List the type of antigen and the type of antibodies present in each ABO blood type. Describe how the presence or absence of Rh antigen results in blood being classified as positive or negative. Distinguish between the development of anti-­‐Rh antibodies and the development of anti-­‐A and anti-­‐B antibodies. Predict which blood types are compatible and what happens when the incorrect ABO or Rh blood type is transfused. State which blood type is considered the universal donor and which blood type is considered the universal recipient, and explain why. 1. Discovery of blood groups: (For a great blood typing game: http://www.nobelprize.org/educational/medicine/bloodtypinggame/) Experiments with blood transfusions, the transfer of blood or blood components into a person's blood stream, have been carried out for hundreds of years. Many patients have died and it was not until 1901, when the Austrian Karl Landsteiner discovered human blood groups, that blood transfusions became safer. Mixing blood from two individuals can lead to blood clumping or agglutination. The clumped red cells can crack and cause toxic reactions. This can have fatal consequences. Karl Landsteiner discovered that blood clumping was an immunological reaction which occurs when the receiver of a blood transfusion has antibodies against the donor blood cells. Karl Landsteiner's work made it possible to determine blood groups and thus paved the way for blood transfusions to be carried out safely. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1930. 2. ABO blood groups: The differences in human blood are due to the presence or absence of certain protein molecules called antigens and antibodies. The antigens are located on the surface of the red blood cells and the antibodies are in the blood plasma. Individuals have different types and combinations of these molecules. The blood group you belong to depends on what you have inherited from your parents. There are more than 20 genetically determined blood group systems known today, but the AB0 and Rh systems are the most important ones used for blood transfusions. Not all blood groups are compatible with each other. Mixing incompatible blood groups leads to blood clumping or agglutination, which is dangerous for individuals. Nobel Laureate Karl Landsteiner was involved in the discovery of both the AB0 and Rh blood groups. 27 AB0 blood grouping system According to the AB0 blood group system there are four different kinds of blood groups: A, B, AB or 0 (null). Blood group A If you belong to the blood group A, you have A antigens on the surface of your red blood cells and B antibodies in your blood plasma. Blood group B If you belong to the blood group B, you have B antigens on the surface of your red blood cells and A antibodies in your blood plasma. Blood group AB If you belong to the blood group AB, you have both A and B antigens on the surface of your red blood cells and no A or B antibodies at all in your blood plasma. Blood group 0 If you belong to the blood group 0 (null), you have neither A or B antigens on the surface of your red blood cells but you have both A and B antibodies in your blood plasma. Rh factor blood grouping system Many people also have a so called Rh factor on the red blood cell's surface. This is also an antigen and those who have it are called Rh+. Those who haven't are called Rh-­‐. A person with Rh-­‐ blood does not have Rh antibodies naturally in the blood plasma (as one can have A or B antibodies, for instance). But a person with Rh-­‐ blood can develop Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies. A person with Rh+ blood can receive blood from a person with Rh-­‐ blood without any problems. Blood group notation According to above blood grouping systems, you can belong to either of following 8 blood groups: A Rh+ B Rh+ AB Rh+ 0 Rh+ A Rh-­‐ B Rh-­‐ AB Rh-­‐ 0 Rh-­‐ Do you know which blood group you belong to? 28 3. Blood typing: A.You mix the blood with three different reagents including either of the three different antibodies, A, B or Rh antibodies. B.Then you take a look at what has happened. In which mixtures has agglutination occurred? The agglutination indicates that the blood has reacted with a certain antibody and therefore is not compatible with blood containing that kind of antibody. If the blood does not agglutinate, it indicates that the blood does not have the antigens binding the special antibody in the reagent. C.If you know which antigens are in the person's blood, it's easy to figure out which blood group he or she belongs to! 4. What happens when blood clumps or agglutinates? For a blood transfusion to be successful, AB0 and Rh blood groups must be compatible between the donor blood and the patient blood. If they are not, the red blood cells from the donated blood will clump or agglutinate. The agglutinated red cells can clog blood vessels and stop the circulation of the blood to various parts of the body. The agglutinated red blood cells also crack and its contents leak out in the body. The red blood cells contain hemoglobin which becomes toxic when outside the cell. This can have fatal consequences for the patient. The A antigen and the A antibodies can bind to each other in the same way that the B antigens can bind to the B antibodies. This is what would happen if, for instance, a B blood person receives blood from an A blood person. The red blood cells will be linked together, like bunches of grapes, by the antibodies. As mentioned earlier, this clumping could lead to death. People with blood group 0 Rh -­ are called "universal donors" and people with blood group AB Rh+ are called "universal receivers." Rh+ blood can never be given to someone with Rh -­ blood, but the other way around works. For example, 0 Rh+ blood can not be given to someone with the blood type AB Rh -­. 5. Blood transfusions? Of course you can always give A blood to persons with blood group A, B blood to a person with blood group B and so on. But in some cases you can receive blood with another type of blood group, or donate blood to a person with another kind of blood group. 29 6. How are blood types inherited? When we talk about inheritance you must distinguish between the phenotype which is what you actually see, in this case blood type and the genotype which is what alleles you inherit. For example: You could have a phenotype of Type A blood, but your genotype could be AA or AO sometimes written Blood Group Antigens AB Rh+ A, B and Rh Antibodies Can give blood to Can receive blood from None AB Rh+ AB Rh+ AB Rh -­ A Rh+ A Rh -­ B Rh+ B Rh -­ 0 Rh+ 0 Rh -­ AB Rh -­ A and B None (Can develop Rh antibodies) AB Rh -­ AB Rh+ AB Rh -­ A Rh -­ B Rh -­ 0 Rh -­ A Rh+ A and Rh B A Rh+ AB Rh+ A Rh+ A Rh -­ 0 Rh+ 0 Rh -­ A Rh -­ A B (Can develop Rh antibodies) A Rh -­ A Rh+ AB Rh -­ AB Rh+ A Rh -­ 0 Rh -­ B Rh+ B and Rh A B Rh+ AB Rh+ B Rh+ B Rh -­ 0 Rh+ 0 Rh-­‐ B Rh -­ B A (Can develop Rh antibodies) B Rh-­ B Rh+ AB Rh-­ AB Rh+ B Rh -­ 0 Rh -­ 0 Rh -­ None A and B (Can develop AB Rh+ 0 Rh -­ Rh antibodies) AB Rh -­ A Rh+ A Rh -­ B Rh+ B Rh -­ 0 Rh+ 0 Rh -­ as IA IA or IAi. If you add in the Rh factor Type A positive blood or a phenotype of A+ could have a genotype of AA++; AA+-­‐;AO++ or AO+-­‐. • Complete the Table 1 at the beginning of the lab report to make sure that this makes sense. 30 7. Blood Typing Exercise Blood typing is done using antibodies to test for the presence of antigens on the surface of the red blood cell. Antigen-­‐Antibody Reactions This exercise allows you to examine the antigen-­‐antibody reactions of known blood types. Each table should take one spot plate and one set of dropper bottles. The bottles are labeled A+, AB-­‐, B+, O+ to represent each of those blood types, and anti-­‐A, anti-­‐B and anti-­‐D (this is a specific type of Rh) to represent the different antisera. Acitivty • Add your anti-­‐sera to the circles on your slides as illustrated below. You only need to add one drop to each circle. Be careful not to let the dropper bottle touch the slide. Anti-­‐ Anti-­‐ Anti-­‐ A B Rh • Now add a single drop of the blood type indicated on the slide to each slide. For example, on the slide • labeled A+, add one drop of the A+ blood sample to each of the circles. Watch for the agglutination reactions. The A and B reactions will produce small clumps. The Rh reaction will form a gel. (This exercise uses simulated blood and antibodies so these observations are not exactly the same as you would observe when typing real blood.) • Now test some known types of blood and fill out Table 2 on the report page to familiarize yourself with the reactions before proceeding to the unknowns. Each lab table will type the blood samples from one of the cases below. You will find the blood samples for the case that you are to do on your lab table. After you have the necessary blood types for your case, compare your samples to the other table that did the same samples and resolve any differences. You will then need to obtain the blood types from the other two cases in order to answer all of the questions. 8. Murder Mystery Party 242: Baby Mama Drama on the High Seas “All aboard!” Captain Baha exclaims over the intercom as a large group of family friends and acquaintances get settled into their quarters for a weeklong cruise on the high seas. Everyone is happy to be on vacation, but none could be more relieved to be on board than Mr. Blue, a recently widowed father. His son, Blue Jr. is accompanying him before heading to his freshman year of college at a top-­‐notch university. In a show of support to her longtime and recently widowed friend, Mrs. Pink is on board along with her daughter Ocean, her daughter’s boyfriend Forrest, and their newborn baby Shyann. A fourth classmate of Blue Jr., Ocean, & Forrest’s also came aboard to let loose on the high seas: Rose. Everything seems to be going swimmingly through dinner, and the group unwinds with a few drinks – that is, everyone but Rose – before the evening activity, the Murder Mystery Party! First Mate Finn gathers everyone in the lounge to explain the rules, then selects a small group of partiers to join him in the adjacent room for further explanation of key roles in the game. Finn chooses Mr. Blue and his son, 31 Rose and Forrest, so Mrs. Pink and Ocean take up two seats at the bar for some mother/daughter bonding. The Murder Mystery Party is underway and the group anxiously awaits the emergence of the key role players from the room with Finn. To the enjoyment of the partiers who believe the game has begun, a cacophany resounds from within the room. An unmistakable gunshot, breaking glass, followed shortly by Rose’s blood-­‐curdling scream and a thud. First Mate Finn stumbles through the doorway clutching what appears to be a knife stuck in his side, and his pistol missing from his holster. The partiers rush to the doorway to find the room covered in blood and wonder if the game has in fact begun, or if they are witness to a true Murder Mystery! Case 1: The Murder Mystery Party Smells Fishy Mr. Blue and Blue Jr. lie in opposite corners of the room, apparently incapacitated. Both Rose and Forrest are missing but are suspected to have exited through the only other door in the room. Thankfully, Dr. Robert is nearby and rushes to Finn’s side to administer emergency treatment. Detective Tracy is also aboard – naturally – and is called to the lounge to investigate. The detective’s forensic analysis reveals that there are only three types of blood in the room. One blood type is Mr. Blue’s, one is Blue Jr.’s, and the other blood type is a trace amount left behind on broken glass from the suspected murderer. Mr. Blue’s blood type: ____________ Blue Jr.’s blood type: _____________ Murderer’s blood type: ______________ Forrest and Rose are apprehended separately by cruise ship security and are questioned by the detective along with the rest of the party regarding the assault on the Blue family. Testimonies provide four suspects. Here’s what Detective Tracy deduced: 1. First Mate Finn was being blackmailed by the Blue family who had discovered that he was really an ex-­‐convict. Finn knew this fact would ruin his chance to ever be made Captain. 2. Blue Jr. may have wanted to keep the blackmail money for himself, and wanted his father out of the way. 3. Ocean suspected that Rose was not drinking because she was in fact pregnant, and furthermore thought that Rose and Forrest may have fooling around behind her back. First Mate Finn’s blood type: ____________ Blue Jr. blood type: ______________ Rose’s blood type: ____________ Forrest’s blood type: ______________ After what seems like forever, Blue Jr. miraculously pulls through, but with a serious concussion and no memory of what transpired in the room. It is suspected that Blue Jr. was knocked out when he was struck by a glass vase, however this could not be confirmed since Finn lost consciousness due to blood loss shortly after emerging from the room. Blood type on the bullet from Finn’s pistol: _____________ Broken glass blood type: ________________ Knife blood type: ________________ 32 Case 1 Conclusion:
1. Mr. Blue was killed by ___________ using the ____________.
2. ____________ struck Blue Jr. with a vase and cut himself/herself.
3. Can it be concluded who stabbed First Mate Finn based on the evidence? Y/N
4. If so, “who dunnit”? ________
Case 2: Who’s Your Daddy? Dr. Robert confirmed that Rose is in fact pregnant and was able to obtain a blood sample from the unborn child. Given Rose’s promiscuity, it has become difficult to determine the paternity of her child. When asked who the father is, Rose defensively turns the spotlight on Ocean, challenging her own faithfulness to Forrest. Extensive blood typing analysis was performed to determine the paternity of both children. The three possible fathers are: Blue Jr., Forrest and Mr. Blue (*Gasp!* Who could it be!??!). Follow the same procedure as you did in Case 1 to type these blood samples to determine if any of these three men may be ruled out as the potential father on the basis of blood type. What are the phenotypes of the two mothers and the three potential fathers? Their possible genotypes? Name Phenotype Possible Genotypes Rose Ocean Forrest Mr. Blue Blue Jr. What are the phenotypes of Shyann and Rose’s unborn child? Possible genotypes? Name Phenotype Possible Genotypes Shyann Rose’s unborn child Case 3: The Exciting Finn-­ale! Between all of Detective Tracy’s sleuthing and interrogations and Dr. Robert’s blood typing analysis, First Mate Finn has lost a lot of blood. His knife wound was stitched up, but the blood loss has been extraordinary and he needs a transfusion to save his life! Fill in the tables below to determine which of the cruise ship passengers would be able to save Finn’s life with a blood transfusion. Name Blood Type Blood types he can receive First Mate Finn Name Blood Type Can he/she donate to Finn? (Y/N) Mr. Blue Blue Jr. Forrest Ocean Rose Ms. Pink 33 Shyann* Detective Tracy AB+ Dr. Robert B+ Captain Baha A-­‐ *Although Shyann has the appropriate blood type, she would not be a viable donor since she is an infant. 34 Attributions: Objectives taken from the HAPS guidelines. Introduction and all figures taken from"Blood Groups, Blood Typing and Blood Transfusions". Nobel Prize (2001). [Blood typing images]. Nobelprize.org. Retrieved July 16, 2012 from http://www.nobelprize.org/educational/medicine/landsteiner/readmore.html. Links to more information: • http://nobelprize.org/educational_games/medicine/landsteiner/ • Blood Typing Examples • Possible parent or child blood types • Blood conversion information 35 PAGE INTENTIONALLY LEFT BLANK
36 Lab 13 Blood Typing Lab Report Name ________________________________ Section______________ Table 1 Complete this table including all possible genotypes. Phenotype Genotype AA++, AA+-­‐, AO+-­‐, AO++ A-­‐ O+ OO-­‐-­‐ AB+ AB-­‐-­‐ B+ B-­‐ Table 2 After testing the known blood types, fill out the table below to ensure that you can see the expected reaction and understand what the reaction means. Blood Type Reacted with Anti-­‐ Reacted with Anti-­‐ Reacted with Anti-­‐ Antigens Present A? (Yes/No) B? (Yes/No) Rh? (Yes/No) on Surface? Name Them Case 1 Mr. Blue’s blood type: ____________ Blue Jr.’s blood type: _____________ Murderer’s blood type: ______________ First Mate Finn’s blood type: ____________ Blue Jr. blood type: ______________ Rose’s blood type: ____________ Forrest’s blood type: ______________ Blood type on the bullet from Finn’s pistol: _____________ Broken glass blood type: ________________ Knife blood type: ________________ Case 1 Conclusion:
1. Mr. Blue was killed by ___________ using the ____________.
2. ____________ struck Blue Jr. with a vase and cut himself/herself.
3. Can it be concluded who stabbed First Mate Finn based on the evidence? Y/N
4. If so, “who dunnit”? ________
37 Case 2 4. What are the phenotypes of the two mothers and the three potential fathers? Their possible genotypes? Name Phenotype Possible Genotypes Rose Ocean Forrest Mr. Blue Blue Jr. 5. What are the phenotypes of Shyann and Rose’s unborn child? Possible genotypes? Name Phenotype Possible Genotypes Shyann Rose’s unborn child 6. Given this information, can any of these men be excluded as Shyann’s father? Why or why not? 7. Given this information, can any of these men be excluded as the father of Rose’s unborn child? Why or why not? 8. Mr. Blue remembered that his wife, Blue Jr.’s mother, had to receive regular blood transfusions and thankfully had the universal receiver blood type, AB+. What is Blue Jr.’s genotype? Mr. Blue’s genotype? Does this provide enough information to exclude Blue Jr. as Shyann’s father? Why or why not? 38 Case 3 Name Blood Type Blood types he can receive First Mate Finn Name Blood Type Can he/she donate to Finn? (Y/N) Mr. Blue Blue Jr. Forrest Ocean Rose Ms. Pink Shyann* Detective Tracy AB+ Dr. Robert B+ Captain Baha A-­‐ *Although Shyann has the appropriate blood type, she would not be a viable donor since she is an infant. 9. Assume Blue Jr. sustained injuries that also required a blood transfusion. Who is the only (reasonable) viable donor? 10. If Blue, Jr. were given Rh+ blood will he have a transfusion reaction? Explain. 11. Another passenger has antibody A in her blood with no Rh antibody. What blood type does she have? 39 PAGE LEFT INTENTIONALLY BLANK 40